Methods and kits used in the detection of fungus

ABSTRACT

The invention encompasses method of using quantitative PCR to detect fungal organisms in clinical and environmental samples to generate standards that allow the quantification of fungal organisms in the samples.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. application Ser. No.13/014,645, filed on Jan. 26, 2011, which claims priority to U.S.Provisional Application Ser. No. 61/298,453, filed on Jan. 26, 2010,both of which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

Generally, the invention relates to molecular methods of detectingmicroorganisms. Specifically, the invention relates to methods ofdetecting a wide variety of fungal species and quantifying the totalamount fungus in a sample.

BACKGROUND OF THE INVENTION

Invasive Fungal Infections (IFI) are an important cause of disease,especially in immuncompromised patients, including patients undergoinghigh dose chemotherapy, patients receiving stem cell and bone marrowtransplants, preterm neonates, intensive care patients, and patientswith acquired or innate immune deficiencies. Fungal, bacterial, viral,helminth, and other infections each require different courses oftreatment and choosing the wrong treatment could cause unnecessary sideeffects and extend patient suffering. Clearly, a clinician needs arapid, single diagnostic test to differentiate fungal infections frominfections with other microorganisms.

Using current techniques, fungal infections are difficult to diagnose inclinical setting because fungi are difficult to culture, with the timeof culture often extending beyond clinical utility and with culturefailures (rendering no useful diagnostic information) frequent. (Preunerand Lion, Expert Rev. Mol. Diagn. 9, 397-401, 2009). This results in anunder-diagnosis and under-treatment of fungal infections.

PCR amplification techniques have been used to detect fungal nucleicacids directly isolated from samples without the need for culture. Thedevelopment of these techniques has been hindered by fungal contaminantsthat inhibit PCR (Borman et al, J Antimicrob Chemother 61 17-112, 2008)or by appropriate experimental controls that would allow the researcherto detect the presence of these or other contaminants (Khot andFredricks Expert Rev Anti Infect Ther 7, 1201-1221, 2009).

A further challenge to the development of PCR assays for fungalinfection is the development of a single, broad range PCR assay thatdetects a wide variety of fungi including a wide range of infectivefungi strains and species, fungi not normally known to infect humanbeings, or even fungi that are not characterized. Such an assay must bebroad enough to amplify the vast majority of known fungal species(including those that are difficult to culture,) but selective enoughthat contaminating human DNA or other contaminating DNA are notsignificantly amplified. Many attempts have been made, but none havedone so using degenerate PCR primers. Additionally, such an assay mustalso be able to quantify the fungal load to provide additional clinicalutility and the ability to create a clonal library for identificationand quantification of individual fungal species.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to primers and primer sets and methods ofuse of those primer sets to detect, identify, and quantify a widevariety of fungi. More specifically the invention incorporates primersets that amplify a region of the 18S rRNA gene of fungi to provide apan-fungal quantitative PCR detection and quantification assay. Thefungi may be detected by a PCR reaction using an oligonucleotide thatincludes SEQ ID NO. 1 as a forward primer and an oligonucleotide thatincludes SEQ ID NO. 3 as a reverse primer. In one embodiment of theinvention, a forward primer and a reverse primer are added to a firstmixture comprising a nucleic acid isolated from a sample. The mixture issubjected to conditions that allow nucleic acid amplification and thepresence or absence of fungi is detected on the basis of a result of thenucleic acid amplification. The forward primer and the reverse primermay either or both be less than 50 nucleotides in length. The method mayfurther comprise the addition of an oligonucleotide probe that includesSEQ ID NO. 2 to the mixture. The oligonucleotide probe may be less than50 nucleotides in length. The oligonucleotide probe may comprise afluorescent label. The fluorescent label may be any fluorescent labelincluding but not limited to: HEX, TET, 5-FAM, 6-FAM, JOE, Cy3, Cy5,ROX, TAMRA, dR110, dR6G, VIC, NED, dROX PET, Gold540, LIZ, and TexasRed. The oligonucleotide probe may also comprise a quencher. Thequencher may be any quencher including TAMRA, BHQ1, BHQ2, BHQ+ orDABCYL. The forward primer may comprise a forward primer mixture thatreflects the degeneracy in SEQ ID NO. 1. The forward primer mixture maycomprise SEQ ID NO. 6 and SEQ ID NO. 7 in any proportion. The reverseprimer may comprise a reverse primer mixture that reflects thedegeneracy in SEQ ID NO. 3. The reverse primer mixture may comprise SEQID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15 in any proportion. Thesample may be any sample derived from a subject such as an animal,including a human being. The sample may also be any sample derived froman environmental source.

The method may further comprise adding a second forward primer thatincludes SEQ ID NO. 4 to a second mixture comprising a nucleic acidisolated from the sample, adding a second reverse primer that includesSEQ ID NO. 5 to the second mixture, and subjecting the second mixture toconditions that allow nucleic acid amplification. The second forwardprimer may comprise a second forward primer mixture that reflects thedegeneracy of SEQ ID NO. 4. The second forward primer mixture maycomprise SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, and SEQ ID NO. 19in any proportion. The second reverse primer may comprise a secondreverse primer mixture that reflects the degeneracy of SEQ ID NO. 5. Thesecond reverse primer mixture may comprise SEQ ID NO. 20, SEQ ID NO. 21,SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO.26, and SEQ ID NO. 27 in any proportion. The second forward primer andthe second reverse primer may each comprise a cloning sequence thatfacilitates cloning of the product into a nucleic acid vector. Thecloning sequence may be any cloning sequence including a restrictionendonuclease site. The method may further comprise ligating the productinto the nucleic acid vector, measuring the copy number of the nucleicacid vector, deleting the nucleic acid vector so as to generate astandard set, and comparing a result of the nucleic acid amplificationof the first mixture to a result of a nucleic acid amplification of athird mixture, wherein the third mixture comprises a standard from thestandard set and thereby quantifying the amount of fungus in the sample.

In another embodiment of the invention, a fungal clone library from asample may be generated by adding a forward primer that includes SEQ IDNO. 4 to the mixture, adding a reverse primer that includes SEQ ID NO. 5to the mixture, and subjecting the mixture to nucleic acidamplification. The forward and reverse primers may each comprise asequence that facilitates cloning into a nucleic acid vector. The methodmay further comprise isolating a product of the nucleic acidamplification and ligating the product into the nucleic acid vector. Thenucleic acid vector may be any vector including a plasmid vector.

In another embodiment of the invention, a kit that facilitates thedetection of fungus in a sample may comprise a first oligonucleotidethat includes SEQ ID NO. 1, a second oligonucleotide that includes SEQID NO. 3, and an indication of a result that signifies the presence offungus in the sample. The kit may further comprise a thirdoligonucleotide that includes SEQ ID NO. 2. The kit may further comprisea fourth oligonucleotide that includes SEQ ID NO. 4. The kit may furthercomprise a fifth oligonucleotide that includes SEQ ID NO. 5. The resultmay be any result, such as a Ct value. The indication may be anyindication, such as a positive control or a written Ct value. If theindication comprises a written indication, it may be provided via theInternet.

The present invention provides among other things: a single-mixtureRTPCR assay capable of identifying the vast majority of fungal species.

It is an object of the invention to provide unbiased fungal discovery.

It is an object of the invention to provide a tool that allows theanalysis of samples without a priori knowledge of the fungi present inthe sample or whether or not there is fungus present in the sample.

It is an object of the invention to improve diagnosis of fungalinfections.

DESCRIPTION OF THE FIGURES

A more complete understanding of the present invention may be derived byreferring to the detailed description when considered in connection withthe following illustrative figures.

FIG. 1 depicts an amplification plot of a standard curve consisting ofthe indicated number of copies of 18S rRNA using SEQ ID NO. 1 and SEQ IDNO. 3 as primers and SEQ ID NO. 2 as a probe. Note that human and mouse18S rRNA amplifies below the dynamic range of the assay

FIG. 2 depicts an amplification plot of three serial 1/10 dilutions of asample of Candida lusitaniae in an assay using SEQ ID NO. 1 and SEQ IDNO. 3 as primers and SEQ ID NO. 2 as a probe.

FIG. 3 depicts an amplification plot of three serial 1/10 dilutions of asample of Candida quercitrusa using SEQ ID NO. 1 and SEQ ID NO. 3 asprimers and SEQ ID NO. 2 as a probe.

FIG. 4 depicts an amplification plot of three serial 1/10 dilutions of asample of Candida tropicalis using SEQ ID NO. 1 and SEQ ID NO. 3 asprimers and SEQ ID NO. 2 as a probe.

FIG. 5 depicts an amplification plot of three serial 1/10 dilutions of asample of Epidermophyton floccosum using SEQ ID NO. 1 and SEQ ID NO. 3as primers and SEQ ID NO. 2 as a probe.

FIG. 6 depicts an amplification plot of three serial 1/10 dilutions of asample of Exophiala dermatiditis using SEQ ID NO. 1 and SEQ ID NO. 3 asprimers and SEQ ID NO. 2 as a probe.

Elements and acts in the figures are illustrated for simplicity and havenot necessarily been rendered according to any particular sequence orembodiment.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, and for the purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the various aspects of the invention. It will beunderstood, however, by those skilled in the relevant arts, that thepresent invention may be practiced without these specific details. Inother instances, known structures and devices are shown or discussedmore generally in order to avoid obscuring the invention. Aspects andapplications of the invention presented here are described below in thedrawings and

If the provisions of 35 U.S.C. §112, ¶6 are invoked to define theclaimed inventions, it is intended that the inventions not be limitedonly to the specific structure, material or acts that are described inthe preferred embodiments, but in addition, include any and allstructures, materials or acts that perform the claimed function asdescribed in alternative embodiments or forms of the invention, or thatare well known presently or later-developed, equivalent structures,material or acts for performing the claimed function.

The present invention comprises a method that uses a set ofoligonucleotide PCR primers that is capable of detecting well over 90%of the known species of fungi in a sample. The present invention furtherencompasses a method that creates a set of standards of known copynumber that may be used to quantify the total fungus present in asample. The primer set comprises a forward primer and a reverse primer.The primers are degenerate primers with multiple nucleotides that may besubstituted at the indicated sites. Therefore, the primers may beprovided as a mixture of primers with different nucleotides substitutedat the indicated sites. Each primer of the primer mixture may be presentin equal amounts or different primers in the primer mixtures may bepresent in unequal proportions. The invention may also encompass the useof an oligonucleotide probe capable of hybridizing to the regionamplified by the forward primer and the reverse primer. Quantitative PCRplatforms may use such a probe labeled with a fluorescent label and aquencher molecule in order to help quantify the amount of the specificnucleic acid present in the sample.

In some embodiments of the invention, the amplification is performedwith a forward primer and a reverse primer intended to amplify theentirety of the 18S rRNA gene of a sample. This is intended to producean amplification product that may be used as either a standard (afterthe copy number of the amplification product is determined) or as alibrary that includes the 18S rRNA from all fungal species in a sample.These primers may also comprise sequences that facilitate cloning of theamplification product into a cloning vector such as restriction enzymesites.

In general, nucleic acid amplification is a process by which copies of anucleic acid may be made from a source nucleic acid. In some nucleicamplification methods, the copies are generated exponentially. Examplesof nucleic acid amplification include but are not limited to: thepolymerase chain reaction (PCR), ligase chain reaction (LCR)self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA,) strand displacement amplification (SDA)amplification with Qβ replicase, whole genome amplification with enzymessuch as φ29, whole genome PCR, in vitro transcription with Klenow or anyother RNA polymerase, or any other method by which copies of a desiredsequence are generated.

Polymerase chain reaction (PCR) is a particular method of amplifyingDNA, generally involving the making of a reaction mixture by mixing anucleic sample, two or more primers, a DNA polymerase, which may be athermostable DNA polymerase such as Taq or Pfu, and deoxyribosenucleoside triphosphates (dNTP's). In general, the reaction mixture issubjected to temperature cycles comprising a denaturation stage(typically 80-100° C.) an annealing stage with a temperature that may bebased on the melting temperature (Tm) of the primers and the degeneracyof the primers, and an extension stage (for example 40-75° C.)

Quantitative PCR incorporates a detectable reporter into the reactionmixture in order to quantify the amount of template amplification. Thedetectable reporter may be, for example, a fluorescent label. The signalfrom the reporter may be detectable upon incorporation into theamplified DNA as is the case with the SYBR Green molecule.Alternatively, the detectable reporter may be linked to anoligonucleotide probe such as in the case of TaqMan™ quantitative PCR.

The oligonucleotide probe may also comprise a quencher molecule. Thequencher hides from detection the majority of the fluorescence that maybe emitted by the fluorescent label when the oligonucleotide probe is insolution. PCR amplification removes the quencher from the probe,rendering the fluorescent molecule detectable. Therefore the quantity orintensity of the fluorescence may be correlated with the amount ofproduct formed in the reaction. One skilled in the art would be capableof calculating the amount of target nucleic acid (either DNA or RNA)present in a reaction mixture comprising a sample from the quantity ofthe change in fluorescence. Examples of fluorescent labels that may beused in quantitative PCR include but need not be limited to: HEX, TET,6-FAM, JOE, Cy3, Cy5, ROX, TAMRA, and Texas Red. An oligonucleotideprobe used in quantitative PCR may also comprise a quencher. Examples ofquenchers that may be used in quantitative PCR include, but need not belimited to TAMRA (which may be used with any of a number of fluorescentlabels such as HEX, TET, or 6-FAM), BHQ1, BHQ2, or DABCYL.

An oligonucleotide probe may include any label. A label may be anysubstance capable of aiding a machine, detector, sensor, device, orenhanced or unenhanced human eye from differentiating a labeledcomposition from an unlabeled composition. Examples of labels includebut are not limited to: a radioactive isotope or chelate thereof, dye(fluorescent or nonfluorescent,) stain, enzyme, or nonradioactive metal.Specific examples include but are not limited to: fluorescein, biotin,digoxigenin, alkaline phosphatese, biotin, streptavidin, ³H, ¹⁴C_(,)³²P, ³⁵S, or any other compound capable of emitting radiation,rhodamine, 4-(4′-dimethylamino-phenylazo)benzoic acid (“Dabcyl”);4-(4′-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)(“Dabsyl”); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid(“EDANS”); Psoralene derivatives, haptens, cyanines, acridines,fluorescent rhodol derivatives, cholesterol derivatives;ethylenediaminetetraaceticacid (“EDTA”) and derivatives thereof or anyother compound that may be differentially detected.

When a nucleic acid such as a primer, oligonucleotide, oligonucleotideprobe or any nucleic acid sequence includes a particular sequence, thesequence may be a part of a longer nucleic acid or may be the entiretyof the sequence. The nucleic acid that includes the sequence may containnucleotides 5′ of the sequence, 3′ of the sequence, or both. The conceptof a nucleic acid including a particular sequence further encompassesnucleic acids that contain less than the full sequence that are stillcapable of specifically hybridizing to the target sequence under anyconditions to which a mixture comprising a nucleic acid may besubjected.

A nucleic acid may be identified by the IUPAC letter code which is asfollows: A—Adenine base; C—Cytosine base; G—guanine base; T or U—thymineor uracil base. M—A or C; R—A or G; W—A or T; S—C or G; Y—C or T; K—G orT; V—A or C or G; H—A or C or T; D—A or G or T; B—C or G or T; N or X—Aor C or G or T. Note that T or U may be used interchangeably dependingon whether the nucleic acid is DNA or RNA. A sequence having less than60% 70%, 80%, 90%, 95%, 99% or 100% identity to the identifying sequencemay still be encompassed by the invention if it is able of binding toits complimentary sequence and/or priming nucleic acid amplification ofa desired target sequence. If a sequence is represented in degenerateform; for example through the use of codes other than A, C, G, T, or U;the concept of a nucleic acid including the sequence also encompasses amixture of nucleic acids of different sequences that still meet theconditions imposed by the degenerate sequence.

Quantitative PCR primers with degenerate sequences may also be suppliedas a primer mixture with nucleotides meeting the conditions set by thedegenerate primer sequence. For example, a primer mixture including SEQID NO. 1 may comprise a mixture of GGAAAACTCACCAGGTCCAG (SEQ ID NO. 6),and GGGAAACTCACCAGGTCCAG (SEQ ID NO. 7). A primer mixture including SEQID NO. 3 may comprise a mixture of GCACTATCCCCAGCACGA (SEQ ID NO. 8),GCACTATCCCCATCACGA (SEQ ID NO. 9), GCTCTATCCCCAGCACGA (SEQ ID NO. 10),GCTCTATCCCCATCACGA (SEQ ID NO. 11), GGACTATCCCCAGCACGA (SEQ ID NO. 12),GGACTATCCCCATCACGA (SEQ ID NO. 13), GGTCTATCCCCAGCACGA (SEQ ID NO. 14).and GGTCTATCCCCATCACGA (SEQ ID NO. 15). A primer mixture including SEQID NO. 4 may comprise a mixture of GGAGAAAGAGCCTGAGA (SEQ ID NO. 16),GGAGAAGGAGCCTGAGA (SEQ ID NO. 17), GGAGAGAGAGCCTGAGA (SEQ ID NO. 18),and GGAGAGAGAGCCTGAGA (SEQ ID NO. 19). A primer mixture including SEQ IDNO. 5 may comprise a mixture of CTAGGAATTCCTCGTTCAAG (SEQ ID NO. 20),CTAGGAATTCCTCGTTGAAG (SEQ ID NO. 21), CTAGGCATTCCTCGTTCAAG (SEQ ID NO.22), CTAGGCATTCCTCGTTGAAG (SEQ ID NO. 23), CTAGGGATTCCTCGTTCAAG (SEQ IDNO. 24), CTAGGGATTCCTCGTTGAAG (SEQ ID NO. 25), CTAGGTATTCCTCGTTCAAG (SEQID NO. 26), and CTAGGTATTCCTCGTTGAAG (SEQ ID NO. 27).

An oligonucleotide includes any DNA or RNA reagent of two or morenucleotides, whether from a natural source, artificially synthesized, orproduced through the use of recombinant DNA technology. A nucleotide isan individual deoxyribonucleotide or ribonucleotide base such as A, C,G, T, or U. An oligonucleotide is often engineered to be capable ofbinding a nucleic acid sequence. An oligonucleotide may be anypolynucleotide of at least 2 nucleotides. Oligonucleotides may be lessthan 10, less than 15, less than 20, less than 30, less than 40, lessthan 50, less than 75, less than 100, less than 200, less than 500, ormore than 500 nucleotides in length. While oligonucleotides are oftenlinear, they may, depending on their sequence and conditions, assume atwo- or three-dimensional structure. Oligonucleotides may be chemicallysynthesized by any of a number of methods including sequentialsynthesis, solid phase synthesis, or any other synthesis method nowknown or yet to be disclosed. Alternatively, oligonucleotides may beproduced by recombinant DNA based methods. One skilled in the art wouldunderstand the length of oligonucleotide necessary to perform aparticular task. Oligonucleotides may be directly labeled, used asprimers in PCR or sequencing reactions, or bound directly to a solidsubstrate as in oligonucleotide arrays.

The invention encompasses methods of identifying fungi through the useof DNA sequencing, such as Sanger sequencing, next generationsequencing, pyrosequencing, SOLID sequencing, massively parallelsequencing, pooled, and barcoded DNA sequencing or any other sequencingmethod now known or yet to be disclosed.

In Sanger Sequencing, a single-stranded DNA template, a primer, a DNApolymerase, nucleotides and a label such as a radioactive labelconjugated with the nucleotide base or a fluorescent label conjugated tothe primer, and one chain terminator base comprising a dideoxynucleotide(ddATP, ddGTP, ddCTP, or ddTTP, are added to each of four reaction (onereaction for each of the chain terminator bases). The sequence may bedetermined by electrophoresis of the resulting strands. In dyeterminator sequencing, each of the chain termination bases is labeledwith a fluorescent label of a different wavelength which allows thesequencing to be performed in a single reaction.

In pyrosequencing, the addition of a base to a single stranded templateto be sequenced by a polymerase results in the release of aphyrophosphate upon nucleotide incorporation. An ATP sulfyrlase enaymeconverts pyrophosphate into ATP which in turn catalyzes the conversionof luciferin to oxyluciferin which results in the generation of visiblelight that is then detected by a camera.

In SOLID sequencing, the molecule to be sequenced is fragmented and usedto prepare a population of clonal magnetic beads (in which each bead isconjugated to a plurality of copies of a single fragment) with anadaptor sequence and alternatively a barcode sequence. The beads arebound to a glass surface. Sequencing is then performed through 2-baseencoding.

In massively parallel sequencing, randomly fragmented targeted DNA isattached to a surface. The fragments are extended and bridge amplifiedto create a flow cell with clusters, each with a plurality of copies ofa single fragment sequence. The templates are sequenced by synthesizingthe fragments in parallel. Bases are indicated by the release of afluorescent dye correlating to the addition of the particular base tothe fragment.

An oligonucleotide may be added to a mixture by any of a number ofmethods including manual methods, mechanical methods, or any combinationthereof. An oligonucleotide may be added to a mixture by adding themixture to an oligonucleotide that is conjugated to a substrate such asin a microarray. One may also add the oligonucleotide to a mixture inwhich the target allele to which the nucleic acid has specificity isabsent.

In some aspects of the invention, an oligonucleotide is bound to asubstrate such as a microarray. Examples of microarrays includeconstructs in which a plurality of single stranded oligonucleotideprobes are affixed to a substrate such as silicon glass.Oligonucleotides with a sequence complementary to an allele are capableof specifically binding to that allele to the exclusion of alleles thatdiffer from the specific allele by one or more nucleotides. Labeledsample DNA may be hybridized to the oligonucleotides and detection ofthe label is correlated with binding of the sample and consequently thepresence of the target nucleic sample. Alternatively, PCR includingquantitative PCR may be performed in an array format.

Any oligonucleotide bound to a substrate may be covalently bound to thesubstrate or it may be bound by some non-covalent interaction includingelectrostatic, hydrophobic, hydrogen bonding, Van Der Waals, magnetic,or any other interaction by which the oligonucleotide may be attached toa substrate while maintaining its ability to recognize the allele towhich it has specificity. A substrate may be any solid or semisolidmaterial onto which a probe may be affixed, attached or printed, eithersingly or in the presence of one or more additional probes or samples.Examples of substrate materials include but are not limited topolyvinyl, polysterene, polypropylene, polyester or any other plastic,glass, silicon dioxide or other silanes, hydrogels, gold, platinum,microbeads, micelles and other lipid formations, nitrocellulose, ornylon membranes. The substrate may take any form, including a sphericalbead or flat surface. For example, the probe may be bound to a substratein the case of an array or a PCR reaction. The sample may be bound to asubstrate in the case of a Southern Blot.

The invention encompasses methods of detecting the presence of fungus ina sample. A sample may be derived from anywhere that a fungus or anypart of a fungus including fungal spores, buds, or hyphae may be foundincluding soil, air, water, solid surfaces (whether natural orartificial,) culture media, foodstuffs, and any interfaces between orcombinations of these elements. A sample may be derived from a subject,such as a plant or animal, including humans. Samples derived fromanimals include but are not limited to biopsy necropsy, or other in vivoor ex vivo collection of prostate, breast, skin, muscle, fascia, brain,endometrium, lung, head and neck, pancreas, small intestine, blood,liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymphnode, bone marrow, kidney, placenta, or fetus. Samples derived fromsubjects may also take the form of a fluid sample such as peripheralblood, lymph fluid, ascites, serous fluid, pleural effusion, sputum,bronchial wash, bronchioalveolar lavage fluid (BALF), cerebrospinalfluid, semen, amniotic fluid, lacrimal fluid, stool, urine, hair, or anyother source of material that may be collected from a living or deadanimal. Samples collected from a plant may be collected from part of aplant or from an entire plant. Samples may be collected by any methodnow known or yet to be disclosed, including swiping or swabbing an areaor orifice, removal of a piece of tissue as in a biopsy, or any methodknown to collect bodily fluids. Samples may be suspected of containing afungus if they are derived from a subject displaying symptoms of afungal infection or from an environmental sample from an area in which afungus is thought to be present.

Examples of fungi that may be detected using the invention include butneed not be limited to: pathogenic fungi such as Candida quercitrusa,Absidia corymbifera, Acremonium strictum, Aspergillus flavus,Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger,Aspergillus versicolor, Aureobasidium pullulans, Candida albicans,Candida famata, Candida guilliermondii, Candida haemulonii, Candidaintermedia, Candida lusitaniae, Candida pararugosa, Candida rugosa,Candida tropicalis, Chaetomium globosum, Coccidioides sp. Corynesporacassiicola, Cryptococcus neoformans, Cunnighamella bertholletiae,Epidermophyton floccosum, Exophiala dermatitidis, Fonsecaea pedrosoi,Fusarium equiseti, Fusarium oxysporum, Fusarium solani, Geotrichumcandidum, Geotrichum capitatum, Malassezia furfur, Microsporum canis,Microsporum gypseum, Neurospora crassa, Paecilomyces lilacinus,Paecilomyces sinensis, Paecilomyces variotii, Penicillium marneffei,Pichia ohmeri, Rhizopus microsporus, Rhizopus oryzae, Rhodotorulaminuta, Rhodotorula mucilaginosa, Rhodotorula slooffiae,Saccharomycopsis crataegensis, Scedosporium apiospermum, Scedosporiumprolificans, Sporothrix schenckii, Stephanoascus ciferrii, Trichophytonmentagrophytes, Trichophyton rubrum, Trichosporon asahii, Trichosporonasteroides, Trichosporon cutaneum, Trichosporon dermatis, Trichosporonfaecale, Trichosporon montevideense, Trichosporon mucoides, andTrichosporon ovoides as well as environmental fungi such as Gigasporagigantea, Acaulospora “brown”, Hebeloma crustuliniformae, Comprinusmicaceous, Sarcosphaera crassa, Pholiota destruens, Pleurotus ostreatus,Cortinarius sp., Helvella queletii, Sclerogaster xerophila, Melanogastermagnisporas, Sedecula pulvinata, Elaphomyces decipiens, Clavulinacristata, Rizopogon sp, Hebeloma crustuliniformae, Tricholoma polulinum,Lactarius sp., Cortinarius sp., Agaricus sp., Xanthomendozagalericulata, Endoconidioma sp., Cladosporium cladosporioides, Phomasp., Cytospora sp., and Alternaria sp.

The invention further encompasses kits containing components thatfacilitate the performance of any of the methods encompassed by theinvention. A kit may be any assemblage or collection of components thatfacilitates a method. A kit that facilitates the invention may includespecific nucleic acids such as oligonucleotides, labeling reagents,enzymes including DNA polymerases such as Taq or Pfu, reversetranscriptase, or any other enzymes and/or reagents that facilitatedetection of the target nucleic acids. Specific nucleic acids mayinclude nucleic acids, polynucleotides, oligonucleotides (DNA, or RNA),or any combination of molecules that includes one or more of the above,or any other molecular entity capable of specific binding to a nucleicacid marker. In one aspect of the invention, the specific nucleic acidcomprises one or more oligonucleotides capable of hybridizing to themarker.

A kit may also contain an indication of a result that indicates aparticular outcome. For example, an indication may be a DNA sequencesignifies the identification of a particular fungal phylum, class,order, family, genus species, subspecies, strain or any otherdelineation of a group of fungi. An indication may be a standard curveconfigured to quantify the amount of fungus present in a sample. Anindication may include a set of standards that is included in the kitthat has been premeasured with regard to copy number. An indication maybe a positive control included with the kit or a set of primers and/orother reagents that may be used to generate the positive control. In thecase of a quantitative PCR reaction, the indication may be a particularCt level or a range of Ct levels. An indication may also include butneed not be limited to: a level of fluorescence or radioactive decay, avalue derived from a standard curve or from a control, or anycombination of these and other outputs. The indication may be printed ona writing that may be included in the kit or it may be posted on theInternet or embedded in a software package.

Elements and acts in the example are intended to illustrate theinvention for the sake of simplicity and have not necessarily beenrendered according to any particular sequence or embodiment. The exampleis also intended to establish possession of the invention by theInventors.

EXAMPLES Example 1

Detection and Quantitation of Fungi by RT-PCR Using Pan-Fungal Primersand Probes

The primers of this invention were designed to enable broad-coverageassays for the detection of fungal organisms. Sequences were exportedfrom the Silva ribosomal RNA database (available at the World Wide Web:arb-silva.de) and used to generate massive multiple sequence alignmentfiles for the 18S rRNA gene, the 5.8S rRNA gene, and the 28S rRNA genefrom all major fungi phyla. The sequence filter setting in the primerdesign software was optimized in order to capture the maximal number ofsequences. A base distribution file was generated from the multiplesequence alignment files by summarizing the number of sequences witheach base (A, T, C, G) at each locus. Regions with at least 6 basesat >99% conservation at the 3′ end of the primer (5′-3′ direction) werethen identified using a combination of Tm assessment and degeneracyminimization (n=3 or less).

After the primer/probe design, specificity of the primer set was checkedby BLAST searching against all human, bacteria, and mouse nucleotidesequences in GenBank using all permutations possible with the resultantprimer sets. Primers with no cross reactivity to human, bacteria, andmouse sequences were selected. A set of degenerate PCR primers and apan-fungal probe targeting the 18S rRNA gene was generated and tested ona variety of fungal isolates by qPCR (see FIGS. 1-6). The forward primerof the primer set includes SEQ ID NO. 1 and the reverse primer includesSEQ ID NO. 3. Either the forward primer or the reverse primer may beless than 100 nucleotides, less than 75 nucleotides, less than 50nucleotides, or less than 30 nucleotides in length. The probe includesSEQ ID NO. 2 and may also be less than 100 nucleotides, less than 75nucleotides, less than 50 nucleotides or less than 30 nucleotides inlength.

TABLE 1  SEQ ID Name Sequence SEQ ID NO. 1 PanFungal_18S_F5′-GGRAAACTCACC AGGTCCAG-3′ SEQ ID NO. 2 PanFungal_18S_prb5′-TGGTGGTGCATG GCCGTT-3′ SEQ ID NO. 3 PanFungal_18S_R 5′-GSWCTATCCCCAKCACGA-3′

Processing of samples for the isolation of fungal DNA for performance ofquantitative PCR is as follows: Liquid specimens are thawed on ice,vortexed for 5-10 seconds, and centrifuged at 8000 rpm for 30 secondsusing airtight bucket swing rotors. This collects sample droplets at thebottom of the tube. 50 μl of RLT buffer (from the Qiagen AllPrepDNA/RNA/Protein Kit) are aliquotted into pre-labelled microtubes. 100 μlof sample are then transferred into the prelabelled microtube, keepingair bubbles to a minimum. Samples are lysed in a Barocycler (PressureBiosciences Inc.) The microtubes are removed and inspected for anyruptures or collapsing. The lysate (approximately 150 μA are then addedto 550 μl of RLT buffer in an Eppendorf tube. This mixture is thencentrifuged for 3 minutes at full speed in an aerosol resistant rotor ina tabletop microcentrifuge at full power. The supernatant is thentransferred to an AllPrep DNA spin column (Qiagen), placed into apre-labelled collection tube, and centrifuged for 30 seconds at 10,000RPM. Then 500 μl of AW1 buffer (Qiagen) is added to the DNA spin column.This is centrifuged for 15 seconds at 10,000 rpm. The flow-through isdiscarded. 500 μl of AW2 is then added to the spin column. Thisassemblage is centrifuged for 2 minutes at full speed. The flow-throughis discarded. The spin column is then placed in a prelabelled 2 mlelution tube. 100 μl of EB is then added to the spin column membrane.This is incubated at room temperature for 2 minutes, centrifuged for 1minute at 10,000 rpm, and DNA is eluted. The eluted DNA is stored at−80° C. when not in use.

The pan-fungal quantitative PCR assay is performed using a master mixcomprising the following concentrations: 1.times. Invitrogen qPCRSuperMix (Invitrogen), 40 μM of the Pan 18S_qPCR forward primer (SEQ IDNO. 1), 40 μM of the Pan 18S qPCR_reverse primer (SEQ ID NO. 3), and 20μM of the Pan 18S qPCR probe (SEQ ID NO. 2). 0.1 μl per reaction ofHi-Di Formamide (Applied Biosystems) is added as well as sufficientultra-pure water to bring the volume of the master mix to 10 μl perreaction. The master mix is then added in 10 μl aliquots to the reactionplate (such as a 384 well plate) and 1 μl of DNA isolated from sample orother DNA template is then added to each well.

A standard curve may be generated by creating a working stock of a knownamount of plasmid standard and diluting that working stock into astandard curve by serial dilutions. For example, a 10⁹ copy per μlworking stock may be diluted into 10⁸, 10⁷, 10⁶, etc. copies per μl anddiluted sufficiently to result in that same number of copies perreaction.

A quantity of 20 ng of non-specific human or mouse genomic DNA may beadded to inhibit binding of the primers to non-specific targets. Theaddition of this non-specific DNA may result in non-specificamplification at Ct values less than 33.

The PCR was run on an Applied Biosystems 7900HT Fast Real-Time PCRsystem. Using this instrument, the conditions were as follows: 3 minutesat 50° C. for UNG treatment, 5 minutes at 95.degree. C. for TaqActivation, then 40 cycles of 15 seconds at 95° C. and 1 minute at 65°C. The results of the quantitative PCR assay described above aredepicted in FIGS. 1-6.

FIG. 1 depicts an amplification plot of a standard curve consisting ofthe indicated number of copies of 18S rRNA using SEQ ID NO. 1 and SEQ IDNO. 3 as primers and SEQ ID NO. 2 as a probe. The target DNA is cloned18S rRNA. Note that human and mouse 18S rRNA amplifies below the dynamicrange of the assay.

FIG. 2 depicts an amplification plot of three serial 1/10 dilutions of asample of Candida lusitaniae.

FIG. 3 depicts an amplification plot of three serial 1/10 dilutions of asample of Candida quercitrusa.

FIG. 4 depicts an amplification plot of three serial 1/10 dilutions of asample of Candida tropicalis.

FIG. 5 depicts an amplification plot of three serial 1/10 dilutions of asample of Epidermophyton floccosum.

FIG. 6 depicts an amplification plot of three serial 1/10 dilutions of asample of Exophiala dermatiditis.

The example shows that the primers of the invention may be used in aquantitative PCR assay that detects a wide variety of fungal species.

Example 2 Ability of 18S rRNA Quantitative PCR Assay to Detect FungalSpecies Relative to Other Pan-Fungal Assays

In silico analysis showed that a qPCR assay using the primer/probe setrepresented by SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 has acoverage of 90.80% of all 2082 available fungal 18S rRNA gene sequences.

In silico validation of pan-fungal primers comprises two parts: a primeranalysis portion and an assay analysis portion. The primer analysis codeparses the primer sequence (accommodating for degeneracies). The primerlocation in the alignment is determined and the alignment is scanned forsequences that have poor data in that region. Once poor qualitysequences are removed, the primer and its reverse complement arecompared for perfect matches to each sequence in the alignment. Matchedsequences and missed sequences are recorded in separate output files andthe data is summarized on-screen prior to termination of the program.

The assay analysis portion scans the output files from multiple primeranalysis files to determine the degree of overlap between variousprimers and compares them to the members of the alignment. Again, thereare separate output files for misses and matches for the whole assay andan additional file that lists the identities of the primer componentsthat matched each member of the alignment. The on-screen output is atally of organisms that matched all assay candidates with respect to thetotal number of organisms in the alignment.

Use of this method to validate primers and probes in the 18S rRNA fungaldetection assay (SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3) revealedthat the forward primer (SEQ ID NO. 1) was capable of recognizing 2011out of 2082 fungal species (96.6%) in the Silva Database that hadsequence data in the region that includes the hybridization site of theforward primer. The reverse primer (SEQ ID NO. 3) matched 1991 out of2076 fungal species (95.9%) in the Silva database that had sequence datain the region that includes the hybridization site of the reverseprimer. The oligonucleotide probe (SEQ ID NO. 2) was capable ofrecognizing 2017 out of 2082 fungal species (96.9%) in the Silvadatabase that had sequence data in the region that includes thehybridization site of the oligonucleotide probe.

Other pan-fungal assays are unlikely to match this level of performance.For example, the forward primer from a published pan-fungal quantitativePCR assay (Einsele et al, J. Clin. Microbiol, 35 1353-1360, 1997)matched only 1779 out of 2078 (85.6%) of the fungal species in the SilvaDatabase that had sequence data in the region that includes thehybridization site of the forward primer. The reverse primer in the samereference matched only 1617 out of 2081 (77.7%) of the fungal species inthe Silva Database (available at the World Wide Web: arb-silva.de/) thathad sequence data in the region that includes the hybridization site ofthe forward primer. The probe in the same reference matched only 1387out of 2086 fungal species (66.5%) accessed in the Silva Database.

This represents a considerable improvement over another broad-coveragefungal qPCR assay Imhof et al, Eur J Clin Microbiol Infect Dis 22,558-560 (2003), which has a coverage of 68.07% of the 2082 availablefungal 18S rRNA gene sequences.

Example 3 Primers Used to Generate a Cloned Standard for Use inQuantitative Validation

A set of primers was generated that is capable of producing quantitativecloned standards. Clone libraries may also be generated from pan-fungalPCR primers capable of amplifying the entire 18S rRNA gene.

TABLE 2  SEQ ID Name Sequence SEQ ID NO. 4 18S_TGEN_library_F15′-GGAGARRGAGCCT GAGA-3′ SEQ ID NO. 5 18S_TGEN_library_R15′-CTAGGNATTCCTC GTTSAAG-3′

The SEQ ID NO. 4 and SEQ ID NO. 5 primer set was used to generate acloned plasmid to be used in quantitative and sensitivity/specificitypreliminary validation. Using the cloned standard, the assay has shown adynamic range of 10.sup.9 copies/ul to 10.sup.2 copies/ul withoutamplifying human genomic DNA in this dynamic range.

Example 4 Sequencing Primers for Use in Identifying Fungal Species

Sequencing primers capable of broad coverage are necessary to assessfungal communities and unknown isolates in that they will enable fungalidentification and characterization without a priori knowledge of thecontents of the community or isolates. Using 18S rRNA and 23/28S rRNAsequences from the Silva database, a base distribution of the 18S rRNAand 23/28S rRNA gene sequences was established. Primers were designed tohybridize to (3′) end of 18S rRNA gene and the (5′) end of the 23/28SrRNA gene. This primer set facilitates nucleic acid amplification acrossthe ITS1/5.85/ITS2 complex, generating a 500-2000 by amplicon that maybe sequenced by any sequencing method.

TABLE 3  SEQ ID Name Sequence SEQ ID NO. 28 ITS_TGEN_F15′-CTTSAACGAGGAAT NCCTAGTA-3′ SEQ ID NO. 29 ITS_TGEN_R1 5′-CATWCCCAAACWACYCGACT C-3′ SEQ ID NO. 30 ITS_TGEN_R3 5′-TACTTGTKYGCTAT CGGTCTC-3′

Such primers may be combined with adapters and/or barcoding sequences.In silico analysis showed that the sequencing primers SEQ ID NO. 28 andSEQ ID NO. 29 or alternatively, SEQ ID NO. 28 and SEQ ID NO. 30 wereable to amplify and characterize a high percentage of the fungal speciestested. The ITS_TGEN_F1 forward primer (SEQ ID NO. 28) matched 1941 outof 2057 (94.36%) fungal species in the 18S rRNA gene and the ITS_TGEN_R1reverse primer (SEQ ID NO. 29) matched 996 out of 1101 (90.46%)sequences in the 23 S/28S rRNA gene. The primer set referenced in Imhofet al, Eur J Clin Microbiol Infect Dis 22, 558-560 (2003) failed toreach this level. The reference's ITS1-R (CAGGAGACTTRTAYACGGTCCAG, SEQID NO. 31) matched only 60 out of 1093 (5.49%) sequences, ITS1-F(CTTGGTCATTTAGAGGAAGTAA, SEQ ID NO. 32) matched 743 of the 1626 (45.69%)sequences and ITS1-F_ext (WTGGTYDYNNAGAGGAAGTAA, SEQ ID NO. 33) matched775 of the 1627 (47.63%) sequences.

All references and materials cited herein are hereby incorporated byreference in their entirety.

1. A method of detecting a fungus in a sample, the method comprising thesteps of: adding a first primer that comprises a sequence of SEQ ID NO.1 to a mixture comprising a nucleic acid isolated from the sample;adding a second primer that comprises a sequence of SEQ ID NO. 3 to themixture; subjecting the mixture to conditions that allow nucleic acidamplification; and detecting the presence of the fungus based on aresult of nucleic acid amplification in the mixture.
 2. The method ofclaim 1, wherein the first primer comprises a first mixture ofoligonucleotides comprising sequences of SEQ ID NO. 6 and SEQ ID NO. 7.3. The method of claim 1, wherein the second primer comprises a secondmixture of oligonucleotides comprising sequences of SEQ ID NO. 8, SEQ IDNO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQID NO. 14, and SEQ ID NO.
 15. 4. The method of claim 1 furthercomprising adding an oligonucleotide probe to the mixture.
 5. The methodof claim 4, wherein the oligonucleotide probe comprises the sequence ofSEQ ID NO.
 2. 6. The method of claim 4, wherein the oligonucleotideprobe comprises a fluorescent label.
 7. The method of claim 6, whereinthe fluorescent label is selected from the group consisting of HEX, TET,5-FAM, 6-FAM, JOE, Cy3, Cy5, ROX, TAMRA, dR110, dR6G, VIC, NED, dROXPET, Gold540, LIZ, and Texas Red.
 8. The method of claim 4, wherein theoligonucleotide probe comprises a quencher selected from the groupconsisting of TAMRA, BHQ1, BHQ2, BHQ+ or DABCYL.
 9. The method of claim1 wherein the sample is derived from a subject.
 10. The method of claim9, wherein the subject is an animal.
 11. The method of claim 1, whereinthe sample is derived from an environmental non-animal source.
 12. Amethod of detecting a fungus in a sample, the method comprising thesteps of: adding a first oligonucleotide mixture to an amplificationmixture, wherein the first oligonucleotide mixture comprisesoligonucleotides comprising sequences of SEQ ID NO. 6 and SEQ ID NO. 7and the amplification mixture comprises a nucleic acid isolated from thesample; adding a second oligonucleotide mixture to the amplificationmixture, wherein the second oligonucleotide mixture comprises at leasttwo oligonucleotides selected from the group consisting ofoligonucleotides comprising sequences of SEQ ID NO. 8, SEQ ID NO. 9, SEQID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14,and SEQ ID NO. 15; subjecting the mixture to conditions that allownucleic acid amplification; and detecting the presence of the fungus.13. The method of claim 12 further comprising adding an oligonucleotideprobe to the mixture.
 14. The method of claim 13, wherein theoligonucleotide probe comprises the sequence of SEQ ID NO.
 2. 15. Themethod of claim 13, wherein the second oligonucleotide mixture comprisesoligonucleotides comprising sequences of SEQ ID NO. 8, SEQ ID NO. 9, SEQID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14,and SEQ ID NO.
 16. The method of claim 13, wherein the sample is derivedfrom an animal.
 17. The method of claim 13, wherein the sample isderived from an environmental non-animals source.
 18. A method ofdetecting a fungus in a sample, the method comprising the steps of:adding a first primer that comprises a sequence of SEQ ID NO. 1 to amixture comprising a nucleic acid isolated from the sample; adding anoligonucleotide mixture to the amplification mixture, wherein theoligonucleotide mixture comprises at least four oligonucleotidesselected from the group consisting of oligonucleotides comprisingsequences of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11,SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, and SEQ ID NO. 15;subjecting the mixture to conditions that allow nucleic acidamplification; and detecting the presence of the fungus.
 19. The methodof claim 18, wherein the second oligonucleotide mixture comprisesoligonucleotides comprising sequences of SEQ ID NO. 8, SEQ ID NO. 9, SEQID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14,and SEQ ID NO.
 20. The method of claim 18, wherein the sample is derivedfrom a source selected from the group consisting of an animal and anenvironmental non-animal source.